Susceptibility to fluconazole of Candida clinical isolates determined by FUN-1 staining with flow cytometry and epifluorescence microscopy

作者：Cidália Pina‐Vaz, Filipe Sansonetty, Acácio Gonçalves Rodrigues, Sofia Costa‐de‐Oliveira, José Martinez‐de‐Oliveira, António F. Fonseca · 发表于：Journal of Medical Microbiology · 年份：2001 · DOI：10.1099/0022-1317-50-4-375 · 被引用次数：37 · 研究领域：Antifungal resistance and susceptibility、Fungal Infections and Studies、Probiotics and Fermented Foods

The susceptibility of clinical Candida isolates to fluconazole was assayed by flow cytometry (FCM) and epifluorescence microscopy (EFM), with FUN-1 staining. In all, 25 clinical isolates of Candida spp. (12 sensitive, 3 dose-dependently sensitive and 10 resistant to fluconazole according to the NCCLS M27-A protocol) were treated with increasing concentrations of fluconazole during 1 or 2 h staining with FUN-1 for 30 min and analysed, respectively, by FCM at 575 nm (FL2) and by EFM. Fluconazole-susceptible strains showed an increased accumulation of FUN-1 in comparison with controls as determined by FCM and a reduced metabolic processing of the probe, confirmed by EFM. Conversely, resistant strains showed decreased FUN-1 staining and were able to process the probe. The fluconazole minimal inhibitory concentrations (MICs) determined by FCM or EFM after FUN-1 staining compared very well with the corresponding values determined by the M27-A protocol, indicating that FUN-1 staining can be used as an alternative to the conventional method. MIC values of resistant strains, with the exception of C. krusei, were lower when treatment with fluconazole followed pre-incubation with 0.1 mM sodium azide, a concentration known to inhibit the activity of efflux pumps. These results show that FUN-1 staining can be used as an alternative and rapid method for the assessment of susceptibility of Candida clinical isolates to fluconazole. Furthermore, the results suggest that resistance of Candida ...